1. Field of the Invention
The field of the present invention relates to the detection of controlled substances, and in particular to the detection of a metabolite of Δ9-tetrahydrocannabinol (“Δ9-THC”). More specifically, the invention relates to the detection of 11-nor-Δ9-THC carboxylic acid (“THCA”) in the oral fluid at the picogram per milliliter (pg/ml) level using mass spectrometry, such as gas chromatography/mass spectrometry/mass spectrometry (“GC/MS/MS”) to screen and/or confirm active cannabinoid use in a test subject.
2. Description of Related Art
Marijuana, a known psychoactive drug, is derived from plants of the hemp family that produce significant amounts of cannabinoids. In particular, the most important cannabinoid is Δ9-THC, which is the major physiologically active constituent of marijuana. Δ9-THC is a controlled substance because it has both sedative and depressant-like effects on the cardiovascular and central nervous systems, as opposed to cannabidiol, a non-psychoactive constituent of marijuana. Through smoking marijuana, Δ9-THC is rapidly absorbed from the lungs into the blood stream and metabolized by the liver to a series of polar metabolites with THCA as the primary metabolite. More specifically, microsomal hydroxylation allylic to the Δ9-THC double bond occurs to form 11-OH-THC. Following this, non-microsomal oxidation to THCA occurs via alcohol dehydrogenase.
The carboxylated compound is then conjugated to the glucuronide metabolite. The glucuronide conjugate is excreted as the major urine metabolite along with about 18 nonconjugated metabolites. The enzymes responsible for formation of THCA and THCA-glucuronide are not known to exist in the oral cavity.
Due to the common abuse of cannabinoids, there is a growing need for non-invasive and rapid tests to detect the presence of these controlled drugs in biological specimens. Currently, the presence of Δ9-THC in biological samples, such as urine, can be detected by a number of techniques such as thin layer chromatography (“TLC”), gas chromatography/mass spectrometry (“GC/MS”), GC/MS/MS, radioimmunoassay (“RIA”), or enzyme immunoassay (“EIA”). The established federal guidelines for initial screening cutoffs of Δ9-THC in urine have been set at a level of 50 ng/ml. In addition, the metabolite THCA has been detected in urine. See, e.g., Wang et al, U.S. Pat. No. 5,264,373, col. 1, lines 36-37; Gustafson et al., Urinary Pharmacokinetics of 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol after Controlled Oral Δ9-Tetrahydrocannabinol Administration, J. Anal. Toxicol., Vol. 28, No. 3, at pp. 160-67 (April 2004) (limit of quantitation of 2.5 ng/ml); Lyons et al., A Comparison of Roche Kinetic Interaction of Microparticles in Solution (KIMS) Assay for Cannabinoids, and GC-MS Analysis for 11-nor-carboxy-delta9-tetrahydrocannabinol, J. Anal. Toxicol., Vol. 25, No. 7, at pp. 559-64 (October 2001) (15 ng/ml cutoff); Chiarotta et al., Analysis of 11-nor-9-carboxy-delta(9)-tetrahydrocannabinol in biological samples by gas chromatography tandem mass spectrometry (GC/MS-MS), Forensic Sci. Int., Vol. 114, No. 1, at pp. 1-6 (October 2000) (urine sample spiked with THCA reported over 5 to 50 ng/ml range); Stout et al., Solid-phase extraction and GC-MS analysis of THC-COOH method optimized for a high-throughput forensic drug-testing laboratory, J. Anal. Toxicol., Vol. 25, No. 7, at pp. 550-54 (October 2001).
The presence of THCA has also been detected in hair. See, e.g., Baptista et al., Hair analysis for delta(9)-THC, delta(9)-THC-COOH, CBN and CBD, by GC/MS-EI: Comparison with GC/MS-NCI for delta(9)-THC-COOH, Forensic Sci. Int., Vol. 128, at pp.68-78 (August 2002); Kintz et al., Testing human hair for cannabis. II: Identification of THC-COOH by GC-MS-NCI as a unique proof, J. Forensic Sci., Vol. 40, No. 4, at pp. 619-22 (July 1995) (0.02-0.39 ng/mg of THCA).
In recent years, there have been many reports concerning the use of oral fluid for drug monitoring for marijuana use. Oral fluid offers some advantages over other types of specimens. For example, oral fluid is readily accessible and its collection is perceived as less invasive than urine specimen collection. Thus, oral fluid requires less privacy intrusion than collection of urine. Further, oral fluid collections can easily be observed, and therefore, the specimen is less susceptible to adulteration or substitution by the donor. While testing for the presence of the parent compound Δ9-THC has been performed with oral fluid, there appears to be only a single report of the detection of marijuana metabolites in oral fluids. In 1992, a review paper by Schramm purported to identify THCA, 11-hydroxy-THC, cannabidiol, together with Δ9-THC in a single saliva specimen after smoking marijuana using HPLC and mass spectrometry, but the methodology was not otherwise reported. See Schramm et al.,Drugs of Abuse in Saliva: A Review, J. Anal. Toxicol, Vol. 16, No. 1, at pp 1-9 (1992). According to Schramm, the presence of the THC and its metabolites was not the result of transfer from the blood because radiolabeled Δ9-THC administered by intravenous injection could not be detected in saliva. Id. (citing R. L. Hawks, The Constituents of Cannabis and the Disposition and Metabolism of Cannabinoids, Natl. Inst. Drug Abuse Res. Monog. Ser. 42: 125-37 (1982)); See also U.S. Pat. No. 6,309,827 to Goldstein et al. (example 8) (discussing how Δ9-THC and its metabolites appear to be sequestered in the buccal cavity during smoking rather than passing from the blood to the oral fluid). Thus, Schramm theorized that the Δ9-THC metabolites purportedly detected in the mouth came directly from the marijuana smoke or metabolism in the mouth such that detection of metabolites in saliva was limited to indication of recent use. However, the published studies performed to date have not confirmed this theory. The conclusion of the Schramm paper—that these metabolites, accumulated in the oral fluid from smoked marijuana by metabolism in the mouth—is not supported by the current data regarding the metabolism of THC. THCA and THCA-glucuronide are liver metabolites, and are not known to be formed in the oral cavity. See Watanabe K, et al., A cytochrome P450 isozyme having aldehyde oxygenase activity plays a major role in metabolizing cannabinoids by mouse hepatic microsomes, Biochem Pharmacol., Vol. 46 No. 3, at pp. 405-11 (August 1993).
Further, subsequent attempts by researchers to detect THCA in the oral fluid were unsuccessful when more sophisticated detection equipment was used than that in Schramm. For example, Huestis and Cone found no evidence by GC-MS (detection limit 0.5 ng/ml) of 11-hydroxy-THC or THCA over a period of seven days following smoked marijuana. See M. A Huestis and E. J. Cone, Alternative Testing Matrices, in DRUG ABUSE HANDBOOK, S. Karch, Ed., CRC Press, Boca Raton, Fla., at pp. 799-857 (1998). Further, Peat reported that THCA could not be detected in the mucosal transudate even when the detection limit was as low as 0.1 ng/ml using a GC/MS/MS assay. See Peat, A Brief Introduction to Oral Fluid Drug Testing (Mar. 27, 2000), available at http://www.4intercept.com/clinicals/brief_intro.html. Researchers theorized that the THCA was so strongly bound to plasma proteins that the compound did not diffuse into the oral cavity. See Kintz et al., Pharmacological Criteria to Use Alternative Specimens for DUI Controls (2000) available at www.vv.se/traf_sak/t2000/804.pdf, Kintz et al., Detection of cannabis in oral fluid (saliva) and forehead wipes (sweat) from impaired drivers, J. Anal. Toxicol., Vol. 24, No. 7, at pp. 557-61 (October 2000).
Researchers continue with attempts to develop assays to detect the presence of THCA in the oral fluid in the ng/ml range. For example, Liang and colleagues have attempted to develop a rapid instrumented fluorescence immunoassay for the detection and quantification of tetrahydrocannibanols in oral fluids using lower detection limits for Δ9-THC and THCA of 1.5 ng/ml and 5.5 ng/ml, respectively. These researchers, however, “spiked” pooled saliva from volunteers to provide the appropriate concentration of drug for testing. See Liang et al, A Rapid Instrumented Fluorescence Immunoassay for the Detection of Tetrahydrocannibanols. Similarly, U.S. Pat. No. 6,309,827 to Goldstein et al., has reported “spiking” oral collection devices with various concentrations of metabolites. Table 5 of the patent shows THCA concentrations around 10 ng/ml. The cutoff for oral fluid detection Δ9-THC and the metabolites using EIA was 50 ng/ml total. Thus, although several attempts at detecting THCA in oral fluid have been made, none have successfully done so in a reliable and repeatable manner.